1. Cell Cycle Regulation
Since the present invention relates to the discovery of compounds capable of binding, and thereby inhibiting the aryl hydrocarbon receptor, it is important to understand its role in effectuating many cytopathic events. To do this, key aspects of cell cycle regulation should be presented.
Over the past few years cell biologists have made remarkable progress in identifying the molecules that drive the cell cycle: the carefully choreographed series of events that culminates in cell division. In doing so they have not only provided a better understanding of one of the most fundamental of the cell's activities, they have also opened a new direction for research aimed at pinpointing the cytopathicity of cancer, AIDS, angiogenesis, and a variety of viral diseases associated with cancer or oncogenesis. The reason for this intriguing convergence is that accumulating data indicates that derangements in the cell cycle machinery may contribute to the pathology of a number of apparently unrelated diseases.
A family of cell division control enzymes termed cyclin-dependent kinase's (CDKs), along with the cyclin proteins, serves to control and coordinate the molecular events of cell division in all eukaryotic cells (Norbury and Nurse, 1992; Draetta, 1990; Draetta et al., 1988; Bartlett and Nurse, 1990). Although twelve CDKs have been described, CDK1 kinase remains the most actively studied because of its central role in the control of cell division in both yeast and animal cells (Draetta, 1990; van den Heuvel and Harlow, 1993; Pines and Hunter, 1990; Norbury and Nurse, 1990).
In normal resting cells CDK1 is not expressed or expressed at very low levels, but concentrations of CDK1 increase as the cell enters and passes through G1 and the G1/S transition. CDK1 concentrations reach maximal levels in the S, G2 and M phases (Loyer et al., 1994). As used herein, the word "expression" refers to the level of active protein of any particular protein; expression of a protein may be affected by a variety of factors including changes in transcription, translation and protein catalysis.
In association with cyclin B, CDK1 is the serine/threonine kinase subunit of M-phase-promoting factor (MPF); active MPF triggers the G2/M transition in species ranging from yeast to humans (Brizuela et al., 1989; Draetta, 1990). Several studies also suggest that CDK1 functions in the control of the G1/S transition and as well as the initiation of mitosis (Furukawa et al., 1990; Krek and Nigg, 1991).
The role of CDK proteins is completely dependent upon their expression and kinase activity in the cell cycle. CDK1 kinase activity during the cell cycle is regulated primarily through post-translational modifications including cycles of phosphorylation and dephosphorylation (Ducommun et al., 1991; Norbury et al., 1991) and interactions with cyclins (Booher and Beach, 1987; Ducommun et al., 1991; Williams et al., 1992). Intracellular compartment translocation has also been demonstrated to regulate the substrate availability of the CDK1 protein (Williams et al., 1992; Pines and Hunter, 1991).
The functioning of CDK1 involves the coordination of all events relating to cell division. In this role CDK1 is the central information processing protein. As the cell moves through the cell cycle, information concerning the activities of the cell are sent to CDK1 and as long as these signals indicate proper functioning of the cell, movement through the cell cycle continues. However, should information sent to CDK1 indicate a problem with the cell (e.g. DNA damage, microtubule disruption) progression through the cell cycle would be halted. The block is imposed on the cell cycle at either the G1/S or G2/M interphase.
An increase in CDK1 expression is seen when the cell transforms into a tumor cell. The cellular expression of CDK1 is governed by exposure to cytokines and hormones; the expression of CDK1 is one signal to the cell to initiate the events of cell division. If the events of cell division are operating normally, CDK1 levels will decrease (through specific proteolytic enzymes) and the cell will re-enter the resting state.
However, if the events of cell division are not functioning normally and CDK1 concentrations in the cell remain elevated, cellular processes will be activated that block in cell at the G1/S interphase of the cell cycle. This block is mediated through the p53 protein and involves the formation of a complex with p21 and CDK1 that inhibits the kinase activity of CDK1. The inhibition of the kinase activity of CDK1 essentially blocks the flow of information from this coordinator of cell division and the cell remains in a G1/S stasis until concentrations of CDK1 can be reduced and the cell enters a resting state.
A number of pathologies have been identified with the over expression of CDK1 protein. Increasing evidence supports the relationship of aberrant CDK1 expression and cancer (Yasui et al., 1993; Pardee, 1989). Over expression of CDK1 was noted in 90% of breast tumor cell lines examined (Keyomarsi and Pardee, 1993), in all 40 human cancer lines studied (Bartek et al., 1993) and in all clinical gastric and colon carcinomas examined (Yasui et al., 1993). Proliferation of vascular endothelial cells is mediated through CDK1 expression (Zhou et al., 1994); such stimulation is associated with angiogenesis and functions in the pathology associated with occlusion of arteries following trauma such as angioplasty (Morishita et al., 1994).
CDK1 is also implicated in HIV-1 envelope-mediated cell death. It has been demonstrated that during HIV-1 mediated cytopathogenicity, CD4+ T cells were killed by a mechanism involving functional CDK1. Inhibition of the tyrosine phosphorylation of CDK1 (a step performed in early G1) resulted in an inhibition of the killing of CD4+ T-cells (Cohen et al., 1993). Such a mechanism may also be involved in the cytopathogenicity of other viral diseases such as hepatitis or herpes.
2. HIV-1
Human immunodeficiency virus-1 (HIV) infection results in the development of acquired immunodeficiency syndrome (AIDS). AIDS is characterized by a compromised immune system attributed to the systemic depletion of CD4+ T lymphocytes (T cells) and unresponsiveness of remaining CD4+ T cells. The level of CD4+ T cells serves as a diagnostic indicator of disease progression. HIV infected CD4+ T cells are known to be directly cytopathic to other CD4+ T lymphocytes and this single cell killing event is initiated via IV envelope protein (gp120/41) interaction with the CD4 molecule. Highly virulent isolates of HIV induce syncytia (defined as &gt;4 nuclei within a common cell membrane), a process associated with rapid loss of CD4+ T cells and disease progression. Syncytia formation requires the proteolytic processing of the gp160 envelope precursor, stable association and cell surface expression of the gp120 and gp41 subunits, CD4 binding, and membrane fusion events that follow CD4 binding. These interactions represent a key mechanism of T cell depletion during progressive HIV infection that can occur when only 1 in 1,000 to 1 in 10,000 lymphocytes are productively infected with virus (Harper, M. E. et al., 1986). This accentuates the importance of gp120/41-mediated single cell killing that can occur during HIV infection.
T lymphocyte cell lines that express unprocessed HIV envelope glycoprotein (HIVenv 2-2) or processed HIV envelope glycoprotein (HIVenv 2-8) have been developed and characterized (Tani, 1993, incorporated herein by reference). The HIVenv 2-2 cell line is unable to process the HIV envelope gene (gp160) into gp120 and gp41 and thus is incapable of killing CD4+ T lymphocytes. The cell line HIVenv 2-8, however, does process the envelope gene into gp120 and gp41 subunits into HIV-like infected T cells. The proper processing of the HIV envelope protein has been shown to induce the killing of CD4+ Jurkat T cells in vitro (Tani, 1993). This methodology mimics CD4+ T lymphocyte depletion that occurs in progressive HIV infection resulting in immune dysfunction.
Co-incubation of CD4+ T lymphocytes with HIVenv 2-8 cells or HIV infected CD4+ cells results in a lethal signal generated via interaction of HIV gp120/41 with the CD4 molecule of target T lymphocytes (Kowalski, 1991; Cohen, 1992; Hivroz, 1993; Heinkelein, 1995; Corbeil, 1996, incorporated herein by reference). The CD4+ cells form syncytia, and undergo a process known as apoptosis or programmed cell death.
Research conducted in the areas of eukaryotic cell cycle control and apoptosis have found that the two events are closely linked. Furthermore, many enzymes and transcription factors that function in promoting cellular growth also appear to participate in cell death. These include c-Myc, p53, cyclin dependent kinases, and cyclin proteins, Bcl-2, and Bax. And as already mentioned, HIV-induced cytopathicity has also been shown to invoke alterations in the phosphorylation state and/or the expression of CDK1 (p34.sup.cdc2) and cyclin B (Colten, 1992; Kolesnitchenko, 1995, incorporated herein by reference). Other stimuli that activate programmed cell death also produce aberrant expression and activation of the cyclin dependent kinases.
3. The Aryl Hydrocarbon Receptor
The Aryl Hydrocarbon (Ah) receptor is an intracellular cytosolic protein found in higher vertebrates in several epithelial tissues. The effects of Ah receptor ligands are known almost entirely in regards to their effects on P4501A1 induction, an enzyme system that metabolizes certain xenobiotics (Landers and Bunce, 1991, incorporated herein by reference). The Ah receptor was discovered by Poland and co-workers and studied first as a high affinity binding protein for aryl hydrocarbons of toxicological importance, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Poland et al., 1976 incorporated herein by reference).
Dioxins or dioxin-like compounds are environmental pollutants produced as unwanted byproducts of common industrial processes such as paper bleaching, incineration and chemical manufacturing.
Dioxins or dioxin-like compounds are a loosely defined family of organochlorine molecules with close structural and chemical similarities. Additionally, these compounds, by virtue of their similar structure and chemistry, share a common mechanism of toxicity. The prototypical dioxin, and the best studied, is 2,3,7,8 Tetrachlorodibenzo-P-Dioxin (sometimes called 2,3,7,8-TCDD or TCDD or dioxin). Besides 2,3,7,8 Tetrachlorodibenzo-P-Dioxin, this group of compounds include not only the dibenzo-p-dioxins, but also dibenzofurans, azobenzenes, dibenzo-ethers, certain polychlorinated biphenyls, certain polyaromatics and other compounds. Toxicity of these compounds is dependent on a planar, polyaromatic structure with lateral halogen substitutions.
The biochemical and physiological basis of dioxin toxicity has been the subject of intense scientific scrutiny. Animals vary in their susceptibility to dioxins and in their symptoms. In guinea pigs, as little as 600 ng per kg produces a lethal wasting syndrome. In humans, toxic responses to dioxin exposure include several proliferative aberrations such as hyperkerotinosis and hyperplasia. Despite much research in the area, the biochemical and physiological events that produce toxicity are poorly understood.
Although the ultimate physiological events that produce toxicity are poorly understood, it is generally agreed that toxicity of these chemically and structurally related dioxin-like compounds is due to their ability, by virtue of their chemical and structural properties, to bind to the intracellular Ah receptor. Although the ability of a compound to be a ligand of the Ah receptor is a requirement for dioxin-like toxicity, these compounds must also be able to promote transformation of the receptor to a DNA-binding form subsequent to ligand binding in order to be toxic. The transformation of the Ah receptor comprises a series of poorly understood events that include dissociation of the inactive receptor from a complex of proteins that include one or more molecules of the chaperonin HSP90, the formation of a new complex that includes HSP90-dissociated Ah receptor plus bound dioxin and the nuclear protein Aryl Hydrocarbon Nuclear Translocator (ARNT), and the binding of the Ah receptor/ARNT complex to specific DNA sequences.
These sequences, called Dioxin-Response Elements (DREs) or Xenobiotic-Response Elements (XREs), lie upstream of the promoter regions of certain genes, the most studied being the P4501A1 gene. The binding of the transformed Ah receptor and associated protein(s) to the DREs enhance transcription of the associated genes. The inappropriate expression of these genes are thought to be the early events in the pleiotropic response to dioxins. It is fundamental that dioxins, in order to be toxic, must be able to both bind to the Ah receptor and transform it into an active form, and that this binding/transformation couplet is the central and only defined biochemical event in the toxic effects of dioxins.
Different dioxin-like compounds, although they share a common mechanism of toxicity, have different toxic potencies that can differ by several orders of magnitude. The toxicity of an unknown mixture of dioxin-like compounds can vary considerably depending on the identity and concentrations of the congeners present. Thus, the concept of Toxic Equivalency Factors (TEFs) and Toxic Equivalence (TEQs) have been advanced by some scientists. TEFs are the fractional toxicity of a dioxin-like compounds compared to the most toxic, prototypical 2,3,7,8-TCDD. Published TEFs are arbitrarily assigned values based on consensus toxicity's in the scientific literature. TEQs are the estimated toxic potential of a mixture of these compounds calculated by adding their respective TEFs with adjustment for their respective concentrations. TEFs and TEQs have been promoted by the EPA in order to facilitate their risk and hazard assessment of these compounds when they occur as mixtures.
The sequence of known events when an agonist or Ah ligand binds to the Ah receptor can be summarized as follows. The Ah receptor in the unbound state is found bound to the chaperonin HSP90 and another poorly understood protein or proteins (Perdew and Hollenback, 1990, incorporated herein by reference). Agonists of the Ah receptor such as TCDD, upon binding to the receptor, alter the receptor (commonly referred to as "transformation") so that the liganded Ah receptor separates from the chaperonin complex, translocates to the nucleus, binds to the ARNT protein, binds to specific DNA sequences upstream of the P4501A1 gene sequence as the Ah receptor: ARNT complex, and enhances transcription of P4501A1.
Antagonists and inhibitors of the Ah receptor have not been well-studied. Research interest has focused on potent, toxic agonists of the Ah receptor such as TCDD. Research interest on antagonists of the Ah receptor has focused on understanding the biochemistry of the Ah receptor, interactions among man-made toxins, and as inhibitors of estrogen-mediated gene expression. Known antagonists of the Ah-receptor include some flavone derivatives (Gasiewicz and Rucci, 1991; and Lu et al., 1995, both incorporated by reference) and synthetic aryl hydrocarbons (Merchant and Safe, 1995, incorporated herein by reference).
Ah receptor agonists and antagonists of plant and dietary origin are known (Kleman et al., 1994; Bjeldanes et al., 1991; and Jellinck et al., 1993, all incorporated herein by reference). Interestingly, these compounds are thought to be anti-carcinogens, tumor promoters, or both, however, mechanisms of action remain unknown.
The biochemical effects of agonists of the Ah receptor are generally thought to be Ah receptor-dependent, that is, the potency of the toxic response-is proportional to their ability transform the Ah receptor (Wheelock et al., 1996 incorporated herein by reference), or induce P4501A1 (Zacharewski et al., 1989 incorporated herein by reference). However, the induction of P4501A1 itself is probably not connected with most of the physiological effects of Ah receptor ligands. Ah receptor ligands can act as anti-estrogenic tumor dependent agents by virtue of the ability of the Ah receptor: ARNT complex to interfere with estrogen receptor-mediated transcription. TCDD effects on both cellular proliferation, and apoptosis may occur via perturbation of intracellular signal transduction systems involved with cellular proliferation and apoptosis, as evidenced with by intracellular protein phosphorylation (Ma, 1992, incorporated herein by reference), induction of protein-tyrosine kinases, and cyclin dependent kinases (Ma and Babish, 1993 incorporated by reference).
The natural function of the Ah receptor is unknown, however, deletion of the Ah receptor results in liver abnormalities and immune system impairment. Furthermore, the identification of any endogenous ligand has remained elusive, and how Ah receptor-mediated signaling interacts with cell cycle and apoptotic control is poorly understood, and a direct connection has not been established.
4. Synergistic Effects with Other Compounds
The present invention concerns the identification of compounds useful in the treatment of cellular cytopathic changes, such as those caused by viral infection or some types of cancers. In that effort, the present application has focused on the use of compounds which have been found to be antagonists of the Ah receptor, capable of preventing the transformation of this receptor to its active form. As already discussed, the efficacy of Ah receptor antagonists has much to do with manipulation of certain proteins in the signal transduction pathways, whose consistent overexpression causes cytopathic changes in cells.
Along this line of investigation, it was believed that other compounds which had demonstrated the ability to inhibit cytopathic changes in cells could be used with Ah receptor antagonists in combinational therapy. This possibility of combinational therapy promised the potential to enhance the down-regulation of specific intracellular proteins thought responsible for observed cytopathic changes, such as CDK1, c-Mos, and cyclin B, thereby inhibiting the proliferation of cells demonstrating cytopathic changes. Combinational therapy would therefore act synergistically to enhance the efficacy of the overall therapy.
A specific compound extracted from the plant Andrographis paniculata, andrographolide, is known to have this type of pharmacological activity. This component of Andrographis paniculata, has been shown to have anti-HIV effects. Moreover, andrographolide has been shown to have anti-viral, anti-neoplastic and hepatoprotective properties. (See the document WO 96/17605, Use of Andrographolide Compounds treat or prevent Pathogenicity of Diseases, incorporated herein by reference). Therefore the impetus to use it in a combinational therapy was a strong one. Additionally, it has been found that while andrographolide has been shown to act, on some of the same proteins, it does not bind the Ah receptor. This discovery, and the use of a combinational therapy with various Ah receptor antagonists, then provides for a broader and potentially more effective therapeutic intervention into viral infections generally, as well as AIDS, some cancers, and other pathological conditions than could be expected with each compound alone.
A tremendous advantage of finding compounds such as Ah receptor antagonists or Andrographolide derivatives with which to treat non-viral cellular targets, c-MOS and the downstream proteins CDK1 and cyclin B1, is that this mode of fighting pathological conditions, in addition to its synergistic effects, substantially eliminates the opportunity for viral mutation and alteration to affect the efficacy of the treatment. The implications of these findings are far reaching.